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Article
From Traditional Breeding to Genome Editing
for Boosting Productivity of the Ancient Grain Tef
[Eragrostis tef (Zucc.) Trotter]
Muhammad Numan 1, Abdul Latif Khan 2, Sajjad Asaf 2, Mohammad Salehin 1, Getu Beyene 3,
Zerihun Tadele 4and Ayalew Ligaba-Osena 1,*
Citation: Numan, M.; Khan, A.L.;
Asaf, S.; Salehin, M.; Beyene, G.;
Tadele, Z.; Ligaba-Osena, A. From
Traditional Breeding to Genome
Editing for Boosting Productivity of
the Ancient Grain Tef [Eragrostis tef
(Zucc.) Trotter]. Plants 2021,10, 628.
https://doi.org/10.3390/
plants10040628
Academic Editor: Francesco Carimi
Received: 8 February 2021
Accepted: 22 March 2021
Published: 25 March 2021
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4.0/).
1
Laboratory of Molecular Biology and Biotechnology, Department of Biology, University of North Carolina at
Greensboro, Greensboro, NC 27412, USA; m_numan@uncg.edu (M.N.); m_salehin@uncg.edu (M.S.)
2Natural and Medical Sciences Research Center, Biotechnology and OMICs Laboratory, University of Nizwa,
Nizwa 616, Oman; abdullatif@unizwa.edu.om (A.L.K.); sajjadasaf@unizwa.edu.om (S.A.)
3Donald Danforth Plant Science Center, St. Louis, MO 63132, USA; GDuguma@danforthcenter.org
4Institute of Plant Sciences, University of Bern, Altenbergrain 21, CH-3013 Bern, Switzerland;
zerihun.tadele@ips.unibe.ch
*Correspondence: alosena@uncg.edu
Abstract:
Tef (Eragrostis tef (Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population
and is currently cultivated in several countries for grain and forage production. It is one of the most
nutritious grains, and is also more resilient to marginal soil and climate conditions than major
cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due
to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during
the growing season. Climate change is triggering several biotic and abiotic stresses which are
expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative
and robust approach in order to improve resilience to diverse types of stresses and increase crop
yields. Traditional breeding has been extensively implemented to develop crop varieties with traits
of interest, although the technique has several limitations. Currently, genome editing technologies
are receiving increased interest among plant biologists as a means of improving key agronomic traits.
In this review, the potential application of clustered regularly interspaced short palindromic repeats
(CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience
in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species
have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system.
This is expected to improve stress resilience and boost productivity, thereby ensuring food and
nutrition security in the region where it is needed the most.
Keywords: CRSIPR-Cas; drought tolerance; Eragrostis tef ; genome editing; stress resilience
1. Introduction
The world population is increasing at an alarming rate, demanding an increase in food
production. The Green Revolution of the 1960s has led to a substantial increase in major
cereal production, but that is unlikely to meet the urgent demand for higher food produc-
tion [
1
] under the current climate scenario. To meet world food demands, the production
of major crops alone is insufficient, as they are less suited to extreme climate and low-
input conditions [
2
]. There is an increasing interest in underutilized crops such as tef
(Eragrostis tef (Zucc.) Trotter); millets, including proso millet (Panicum miliaceum Mill.) and
finger millet (Eleusine coracana Gaertn.); and quinoa (Chenopodium quinoa Willd.), which
are more versatile due to their resilience to marginal growing conditions, and outstanding
nutritional values. Despite its valuable traits, the grain yield of tef is very low. In 2018,
the average yield of tef in Ethiopia was only 1.7 ton ha
−1
as compared to maize (4 ton ha
−1
)
and wheat (2.7 ton ha
−1
) [
3
]. Tef is a cereal crop originating in the Horn of Africa, which is
Plants 2021,10, 628. https://doi.org/10.3390/plants10040628 https://www.mdpi.com/journal/plants
Plants 2021,10, 628 2 of 19
widely cultivated in Ethiopia and Eritrea. In Ethiopia, tef is a staple food for about 70% of
the population. The crop is annually cultivated on 2.9 million hectares of land, producing
about 4.5 million tons of grain [
4
]. Tef is tolerant to marginal soil and unfavorable climate
conditions, which makes it a potential crop for arid and semiarid areas as well as poorly
drained soils [
5
]. Tef is also one of the most nutrient-dense crops, containing high amounts
of macro- and micro-nutrients (primarily calcium and iron), amino acids and vitamins [
6
].
Tef cultivation in Ethiopia and around the globe has increased in recent years due to its
many health-related benefits. Since the absence of gluten epitopes has been confirmed
in tef by antibody assays [
7
], it has been recommended as an alternative diet for people
suffering from celiac disease, the immune reaction to consuming gluten containing foods
such as wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and rye (Secale cereal L.),
which affects 0.6–1.0 percent of the population globally [
8
,
9
]. In addition to the extensive
use of tef grain for human consumption, the straw of tef is more nutritious and palatable
as a livestock feed compared to the straw from cereals such as barley and wheat [
10
].
Moreover, tef straw is used as construction material because it serves as an organic binder
for mud used for plastering walls for local houses [
11
]. Various agronomic traits, such
as panicle architecture, tilling, grain size and plant height, have been targets for the im-
provement of tef yield. Grain yield is a highly complex trait which has several components,
including seed weight, form and size of panicles, florets per panicle and number of fertile
tillers [
12
,
13
]. Other important traits that determine grain yield include shoot biomass,
panicle weight and the number of tillers in a plant [
14
]. Furthermore, certain agronomic
traits such as shattering proneness, lodging tolerance, dry matter yield, leaf area, and plant
height directly or indirectly influence grain yield in crops [15,16].
The main factors causing yield loss in tef include susceptibility to lodging, weed
competition, drought, small grain size and soil acidity [
5
]. Although tef shows several
agronomic and nutritionally desirable traits, it is under tremendous pressure due to harsh
environmental stress conditions [
5
]. The crop is relatively resistant to diseases and insect
pests as compared to other cereal crops. Among abiotic stressors, tef yield is significantly
reduced by drought and soil acidity. Weed competition has broad about a range of effects
on the yield of tef in Ethiopia [
17
]. Many direct and indirect strategies of weed control
are employed by farmers [
18
]. Hand weeding and frequent tillage are the two commonly
used methods of weed control in tef production. Furthermore, weeds can be controlled
by herbicide application with proper management of spray times and frequency. How-
ever, the herbicides must be specific to broad-leaved weeds to avoid damaging tef plants.
Taken together, hand weeding, the use of herbicides and resistant tef varieties are viable
alternatives in order to overcome yield loss due to weeds. With proper weed control
methods, improved tef varieties such as Kora and Quncho have been shown to produce
higher yields [19].
Drought is a major abiotic stress which has significant effects on crop yield in most
African countries. Water scarcity has resulted in a fragile ecosystem in Africa’s arid and
semiarid regions. In sub-Saharan Africa, about 1.1 billion people live in drier environments;
however, this number is expected to double by 2050, and is expected to reach 2.5 billion
people [
20
]. Drought stress after planting [
21
] and during the flowering and grain filling
stage has serious effects on crop yields, and up to 60% of yield loss has been reported
in pearl millet at these stages [
21
,
22
]. In tef, drought has been reported to cause about 40%
yield loss [23].
The other major cause of low productivity in tef is lodging, which is the displacement
of the stalks from the vertical position due to wind and rain [
24
]. Lodging occurs frequently
before grain maturity, significantly reducing the grain yield [
25
]. Tef is primarily susceptible
to stem lodging [
26
,
27
]. Panicle length is also associated with lodging tolerance [
25
]. Semi-
dwarf varieties of tef are lodging-tolerant and produce higher yields than tall varieties [
28
].
Lodging limits the use of inputs such as N-fertilizers, exacerbating the susceptibility of
the plant to lodging [29].
Plants 2021,10, 628 3 of 19
To overcome the effects of the constrains mentioned above and to improve the tef
productivity, it is important to develop resistant and high-yield verities. There are several
approaches to increasing crop productivity as well as stress tolerance in crops. Among
these strategies, genome editing techniques have recently received increased attention.
Previous studies have suggested that the productivity of many cereal crops such as
maize [
30
,
31
], rice [
32
–
35
], wheat [
30
,
36
] and other monocots [
37
,
38
] have been improved
using the clustered regularly interspaced short palindromic repeats (CRISPR) system.
In rice (Oryza sativa L.), CRISPR-associated proteins (CRISPR-Cas) systems have been used
to improve tolerance to drought [
39
], cold [
40
] and salt stress [
41
,
42
], ultimately boosting
productivity [
39
]. In wheat, two efficient and simple CRISPR-Cas methods have been de-
veloped to improve productivity and stress resilience [
43
–
45
]. The CRISPR-Cas technology
used in these monocots is expected to be transferred to tef. Therefore, the aim of this review
is to highlight the potential of CRISPR-Cas-mediated gene-editing in trait improvement
in tef.
2. Mechanisms of Tolerance to Lodging and Environmental Constraints in Tef
2.1. Lodging Tolerance
Lodging is the process by which cereal shoots are displaced from an upright position
to a horizontal position [
46
]. Lodging is considered a complex phenomenon, influenced
by several factors, such as diseases, agronomic practice, crop history, soil type, landscape,
geography, rain and wind [
47
]. Stem lodging is the bending or breaking of stem internodes
(lower culm internodes), whereas root lodging is the failure of the root to maintain its in-
tegrity in the soil [
48
]. The application of fertilizers aggravates lodging, and hence the yield
potential of tef. Lodging stress can be reduced by controlling/decreasing plant height.
However, reducing plant height by inhibiting plant growth regulators or introducing
dwarfing genes could lead to crop yield reductions [
47
]; hence, researchers have suggested
targeting traits other than plant height to reduce yield loss due to lodging. A recent study
by Merchuk-Ovnat, et al. [
49
] suggested that early lodging is likely caused by a rapid
increase in inflorescence weight [
49
]. This group also observed variations among the tested
tef population in terms of lodging time and strength, with some populations possessing
the strength to hold the inflorescence in the grain filling season up to a certain point before
they were bent to the ground. Due to its weak stem, tef has high chance of succumbing to
lodging due to rain or wind [
50
]. Modification of the stem’s chemical composition, such as
its cellulose, lignin, structural carbohydrate and silica composition, is expected to increase
lodging-, disease-, and pest-resistance [
51
]. Silicon (Si) is a beneficial plant nutrient that has
been shown to increase tolerance to lodging, diseases and pests, as well as to abiotic stresses
such as drought, salinity, heavy metal stresses, and extreme temperature in various crops,
ultimately leading to increased grain yield [
52
–
56
]. We recently performed greenhouse
experiments to study whether tef benefits from Si application. Our findings revealed that
Si improves grain and biomass yield, stress resilience, and regulates the expression of
Si-transporter genes in tef [
57
]. However, conclusive evidence showing the mechanism of
silicon-induced stress resilience is lacking [58].
Although lodging is the main cause of low yield in tef [
59
], both physiological and
molecular aspects are understudied, and biotechnological, molecular and breeding tech-
niques [
47
] are not well developed to prevent lodging. A partnership formed by the ‘Tef
Improvement Project’ has recently developed semi-dwarf and lodging-tolerant tef varieties,
which are currently being disseminated in farmer’s fields in Ethiopia [60].
Lodging tolerance has been shown to be improved by modulating the biosynthesis
of plant growth regulators (PGRs). For example, the inhibition of gibberellic acid (GA)
has been shown to reduce plant height [
46
,
61
] and decrease lodging susceptibility. Shorter
internodes are associated with reduced plant height [
62
]. During the Green Revolution of
the 1960s and 1970s, inhibition or alteration of GA in rice and wheat was mainly targeted
for developing semi-dwarf varieties, which ultimately boosted the yield of these crops [
63
].
In tef, mutation in the
α
-Tubulin gene is associated with agronomically important traits
Plants 2021,10, 628 4 of 19
such as semi-dwarfism and lodging tolerance [
59
]. Blösch, et al. [
25
] have reported that
panicle angle contributes to lodging tolerance in tef. Jifar, et al. [
28
] also identified some
lodging tolerance genotypes (RIL-91,RIL-244 and RIL-11).
Genes associated with dwarfism in plants have been widely studied [
64
–
69
]. The two
prominent genes of the 1960s Green Revolution were the semi-dwarf (SD1) gene
in rice
[66,70,71]
and reduced height-1 (RHT-B1b and RHT-D1b) in wheat [
72
]. SD1 belongs
to the gibberellin biosynthetic pathway, whereas RHT is a GA response regulator and is
aDELLA protein family gene. DELLA proteins are important components of the signal
transduction pathway of GA, encoded by the wild-type allele of RHT-B1b and RHT-D1b [
73
].
In the Green Revolution wheat varieties, introduction of a stop codon in the N-terminus of
the two reduced height-1 (RHT-B1 and RHT-D1) loci was responsible for the semi-dwarf
and lodging tolerance traits [
72
]. In rice, the enzyme gibberellin 20-oxidase (GA20) encoded
by the SD1 gene is responsible for the biosynthesis of GA [
65
,
74
]. A frame shift mutation
due to a 383-bp deletion in the sd1 allele has been shown to greatly reduce the level of
GA20 oxidase [
66
]. Mutation of the sd1 and RHT homologs in tef could potentially lead
to lodging tolerance and significantly improve grain yield. Similarly, genetic loci (DW1,
DW2,DW3 and DW4) that control plant height across several environmental conditions
have been identified in sorghum. Recently, scientists have transferred these mutations
into a single sorghum line and managed to release a semi-dwarf commercial variety that
contains mutations in three loci (DW1,DW2 and DW4) [
75
,
76
]. This suggests that these
mutations could also be introduced into tef to develop semi-dwarf varieties with improved
stress tolerance and enhanced grain yield.
2.2. Drought Tolerance
Understanding the degree of stress tolerance in crop plants is important in devising
alternative strategies for improving yield and quality. Drought is one of the most important
abiotic stresses affecting plant growth and development. Plants have developed various
mechanisms of drought tolerance [
77
,
78
]. The mechanisms that have been reported in tef
include modifications of stomatal conductance, osmotic adjustment, development of a deep
rooting system and maintenance of cell membrane stability [
79
,
80
]. Development of
a deep root system and osmotic adjustment are major drought stress tolerance mechanisms
in many crops, including tef [
79
]. The association of plant height, root depth and thickness
to drought stress tolerance was previously reported in tef [
79
]. Recently, crosstalk between
plant height and drought tolerance was reported from a study on tef and other small cereals
where semi-dwarf plants were found to be drought-tolerant [
81
]. Osmotic adjustment is
also known to enable tef leaves to maintain leaf turgor pressure (LTP) [
79
,
82
] under extreme
drought conditions by retrieving and absorbing water even from dry soils. Modification of
root growth parameters in response to water scarcity is another strategy used to mitigate
drought stress [
83
,
84
]. For example, the increase in root length of cowpea, peanut and
soybean plants when exposed to drought enabled them to absorb deep soil water [
84
].
Similarly, developing deep-rooted tef plants with an extensive and broad root system is
a desirable trait to withstand drought stress [79].
2.3. Weed Competition and Herbicide Tolerance
Weed competition is another important plant trait in areas of low-input integrated
weed management systems [
85
]. The competitive ability of crops has been divided into
two broad categories; the first category is the crop’s ability to reduce competitor fitness,
whereas the second is the crop’s ability to resist yield losses and withstand its neighbor’s
competitive impact [
86
]. Different terms have been used for these aspects in the literature,
such as “tolerance ability” and “suppressive ability” [87,88].
In Ethiopia, smallholder farmers have adopted some cultural methods to mitigate
the impact of weed competition. Hand weeding and frequent tillage are common practices
used to control weeds in tef production [
17
]. Herbicides are not widely used, mainly due to
economic reasons and shortages of supplies. An alternative strategy in weed management
Plants 2021,10, 628 5 of 19
is the use of cultivars with competitive ability due to their sustainability [
88
,
89
]. However,
information on tef varieties with high weed competitive ability is limited as compared
to other cereals such as oats (Avena sativa L.), barley (Hordeum vulgare L.) and wheat
(Triticum aestivum L.) [
86
]. Tef varieties can be improved using genetic modification tools
such as the CRISPR system to improve weed tolerance and enhance productivity. Potential
genes for weed resistance and yield improvement can be overexpressed in tef or engineered
through the CRISPR-Cas system to minimize the impact of weed competition.
Herbicide-resistant varieties have been developed in crops such as soybean by tar-
geting key genes in amino acid synthesis or other functions. Among these genes, aceto-
lactate synthase (ALS) is involved in the synthesis of branched-chain amino acids such as
isoleucine, leucine and valine [
90
]. ALS is the target site for five non-competitive inhibitor
families—sulfonylaminocarbonyltriazolinones, pyrimidinylthiobenzoates, triazolopyrim-
idines, imidazolinones and sulfonylureas [
91
]. Plants engineered in the ALS gene are
resistant to non-selective herbicides, whereas all non-engineered plants, including weeds,
are sensitive to the non-selective herbicides. A similar principle was implemented to
develop glyphosate-resistant plants in which the EPSPS (5-enolpyruvylshikimate-3-phosphate
synthase) gene was targeted. The EPSPS gene is involved in the shikimate cycle [
92
]. Overex-
pression or knockout of the above-mentioned genes might contribute towards developing
tef plants with resistance to non-selective herbicides.
2.4. Panicle Architecture
Panicle architecture and grain size are important yield traits in cereal crops such as
rice, wheat and barley [
93
–
95
]. There is a direct relationship between agronomic traits
such as panicle number, number of spikelets in panicle, spikelet filling percentage, grain
size and number and crop yield [
96
]. For example, in rice, higher grain yield in a hybrid
variety is associated with the number of spikelets in a panicle [
96
,
97
]. In some crops, genes
that control panicle number and grain size have been identified and modified to increase
yield [
98
–
100
]. For example, OsSPL14 (squamosa promoter binding protein-like 14) gene
and microRNA “OsmiR397” promoted panicle branching and increased grain size in rice,
which ultimately lead to high grain yield [
99
,
101
]. In tef, homologs of these genes remain
to be identified and characterized to determine their role in increasing grain size and to
improve yield.
3. Status of Tef Improvement
3.1. Traditional Breeding: Past and Current Status of Tef Improvement
Scientific research on tef started in Ethiopia in 1950s [
102
]. Early breeding work fo-
cused on germplasm enhancement through collection, characterization, evaluation and
conservation, as well as genetic improvement in which pure lines were selected from
already existing germplasm [
11
,
103
] (Figure 1). Since flower opening characteristics were
revealed in tef in 1974, [
104
], hybridization was used as a means of tef improvement.
Molecular approaches in tef including marker development, genetic linkage maps, genetic
and molecular diversity analysis were initiated during 1995–1998 [
11
]. Further progress
was made during 1998–2003, including the initiation of interspecific hybridization,
in vitro
culture and mutagenesis in order to improve disease and lodging resistance. Over the last
two decades, there has been progress in the area of tef genetic architecture and genomics re-
search [
105
,
106
] (Figure 1). From a total of 42 improved tef varieties released by the National
Research Program in Ethiopia, 18 were developed using the hybridization technique [
107
].
Plants 2021,10, 628 6 of 19
Plants 2021, 10, x FOR PEER REVIEW 6 of 19
Figure 1. Improvement of tef varieties over the last 50 years. The improvement of tef started back
in 1970s with tissue culture techniques, followed by hybridization, the study of molecular diver-
sity, molecular marker analysis, the development of resistant varieties by interspecific hybridiza-
tion and mutation and the recently emerged clustered regularly interspaced short palindromic
repeats (CRISPR)-associated proteins (CRISPR-Cas) genome editing technique. Note: (The pictures
used in this figure were either taken in the author’s labs or drawn using ChemBioDraw software).
3.2. Molecular Marker Development
The application of molecular markers in tef improvement was initiated during 1995–
1998 [11]. Molecular markers near target genes are utilized for marker-assisted selection
(MAS) or marker-assisted breeding (MAB) [108]. They enable the effective use of alleles
during the selection of phenotypes. The most commonly used markers are microsatellites
(simple sequence repeats; SSRs), amplified fragment length polymorphism (AFLPs) and
single nucleotide polymorphisms (SNPs) [108]. During the selection of molecular markers,
some important factors are considered, such as the quality and quantity of required DNA,
procedures for marker assays, the level of polymorphism and the cost of the marker [109].
In tef, the SSRs and expressed sequence tag (EST), restriction fragment length polymor-
phisms (RFLPs) and random amplified polymorphic DNA (RAPD) have been developed
[110,111]. Through SSR analysis, Abraha, et al. [112] identified and improved some im-
portant traits in tef, including grain yield, days to maturity, panicle length and plant
height. Similarly, variability in tef accessions was identified using AFLP markers, which
can be used in seed multiplication and breeding programs [113]. Application of these
Figure 1.
Improvement of tef varieties over the last 50 years. The improvement of tef started back
in 1970s with tissue culture techniques, followed by hybridization, the study of molecular diversity,
molecular marker analysis, the development of resistant varieties by interspecific hybridization
and mutation and the recently emerged clustered regularly interspaced short palindromic repeats
(CRISPR)-associated proteins (CRISPR-Cas) genome editing technique. Note: (The pictures used
in this figure were either taken in the author’s labs or drawn using ChemBioDraw software).
3.2. Molecular Marker Development
The application of molecular markers in tef improvement was initiated during 1995–
1998 [
11
]. Molecular markers near target genes are utilized for marker-assisted selection
(MAS) or marker-assisted breeding (MAB) [
108
]. They enable the effective use of alleles
during the selection of phenotypes. The most commonly used markers are microsatel-
lites (simple sequence repeats; SSRs), amplified fragment length polymorphism (AFLPs)
and single nucleotide polymorphisms (SNPs) [
108
]. During the selection of molecular
markers, some important factors are considered, such as the quality and quantity of re-
quired DNA, procedures for marker assays, the level of polymorphism and the cost of
the marker [
109
]. In tef, the SSRs and expressed sequence tag (EST), restriction fragment
length polymorphisms (RFLPs) and random amplified polymorphic DNA (RAPD) have
been developed [
110
,
111
]. Through SSR analysis, Abraha, et al. [
112
] identified and im-
proved some important traits in tef, including grain yield, days to maturity, panicle length
and plant height. Similarly, variability in tef accessions was identified using AFLP markers,
which can be used in seed multiplication and breeding programs [
113
]. Application of
Plants 2021,10, 628 7 of 19
these markers could play a great role in environmental stress tolerance in tef for improved
productivity. Targeting induced local lesions in genomes (TILLING) is another genetic
method used to identify small deletions or single base pair changes (mutation detection)
in specific target genes [
114
]. In tef, targeting induced local lesions in genomes (TILLING)
was used for targeting and improving valuable agronomic traits such as drought tolerance,
seed size and dwarfism [115].
4. Potential of Genome Editing Technologies for Tef Improvement
Genome editing is one of the most recently developed technologies that has great po-
tential to improve abiotic stress tolerance and boost productivity in tef. In a given genome,
DNA can be replaced, inserted or deleted at an endogenous loci through a robust genetic en-
gineering technique using sequence-specific nucleases (SSNs) [
116
]. SSNs such as CRISPR
and CRISPR-associated protein 9 (CRISPR-Cas9) [
117
–
120
], transcriptional activator-like
effector nuclease (TALEN) [
121
–
123
] and zinc finger nuclease (ZFN) [
124
,
125
] have been
implicated in rapid genome editing in recent years. In addition to these, plant scientists use
other techniques such as base editing, prime editing [
126
] and CRSIPR-Cpf1 [
127
]. Recently,
CRISPR-Cpf1 has successfully used the prime genome editing in wheat Lin, et al. [
128
]
and rice Lin, et al. [
128
], Li, et al. [
129
] genomes. These genome editing tools have been
used in model plants, but with advances in genome editing, these procedures are now cus-
tomized for wide variety of plant species and are usually specific to genotype [
130
] (Figure
2). However, to adopt advanced genetic engineering technologies in tef, there must be
a well-established transformation and regeneration system, which is currently underdevel-
oped or non-existent for underutilized crops including tef. Recent advances in transgenic
technologies have revealed promising tools for enhancing transformation and regeneration
of transgenic lines. For example, overexpression of the maize embryogenic regulator genes
baby boom (Bbm) and Wuschel2 (Wus2) has been shown to produce high transformation
frequencies in numerous previously non-transformable monocot species, including maize
inbred lines, sorghum (Sorghum bicolor (L.) Moench), sugarcane (Saccharum officinarum L.)
and indica rice (Oryza sativa ssp. indica) [
131
]. More recently, Debernardi et al. [
132
] re-
ported that expression of a fusion protein combining wheat growth-regulating factor 4
(GRF4) and its cofactor GRF-interacting factor 1 (GIF1) has been shown to substantially
increase the efficiency and speed of regeneration in wheat, triticale and rice and increase
the number of transformable wheat genotypes. These approaches have great potential for
genetic improvement of tef and other recalcitrant economically important crops.
Since its first application as a plant genome editing technique [
120
,
133
,
134
], CRISPR-
Cas has been widely applied in crop improvement programs [
135
,
136
]. Major crops
that have benefited from the CRISPR-Cas technique include rice [
32
–
35
], maize [
30
,
31
],
wheat [
30
,
36
] and other monocots [
38
]. In rice (Oryza sativa), the CRISPR-Cas system
has been used to enhance drought [
39
], cold [
40
] and salt [
41
,
42
] tolerance, and to boost
productivity [
39
]. Recently, in wheat, which is one of the plant species that is considered
recalcitrant to genetic transformation via the Agrobacterium method, two efficient and simple
CRISPR-Cas methods were developed [
43
–
45
]. Taken together, CRISPR-Cas technology
has been widely implemented in both monocots and dicots, and has great potential to
be implemented in tef improvement so that the performance of the crop against diverse
environmental stresses will be enhanced, with the ultimate goal of boosting productivity.
Plants 2021,10, 628 8 of 19
Plants 2021, 10, x FOR PEER REVIEW 8 of 19
Figure 2. A schematic view of genome editing by zinc finger nuclease (ZFN) and transcriptional
activator-like effector nuclease (TALEN) in tef. A desired gene is selected from tef and integrated
with ZFN and TALEN and then transferred to a cell through a vector, which will then introduce a
break into the double-stranded DNA and integrate the gene of interest into the host genome.
Transformed cells are used to regenerate to whole plants. (Note: the pictures used in this figure
were either taken in the author’s labs or drawn using ChemBioDraw software).
Candidate Tef Genes for CRISPR-Cas Technology
The CRISPR-Cas system has proven efficient because it uses a single guide RNA
through pairing of DNA targeting [137,138]. Targeting of DNA is essential for genome
editing across all organisms [139]. In order to edit any plant gene using the CRISPR-Cas
system, it is not necessary to integrate into the genome. For example, a guide RNA and
Figure 2.
A schematic view of genome editing by zinc finger nuclease (ZFN) and transcriptional
activator-like effector nuclease (TALEN) in tef. A desired gene is selected from tef and integrated with
ZFN and TALEN and then transferred to a cell through a vector, which will then introduce a break
into the double-stranded DNA and integrate the gene of interest into the host genome. Transformed
cells are used to regenerate to whole plants. (Note: the pictures used in this figure were either taken
in the author’s labs or drawn using ChemBioDraw software).
Candidate Tef Genes for CRISPR-Cas Technology
The CRISPR-Cas system has proven efficient because it uses a single guide RNA
through pairing of DNA targeting [
137
,
138
]. Targeting of DNA is essential for genome
editing across all organisms [
139
]. In order to edit any plant gene using the CRISPR-Cas
system, it is not necessary to integrate into the genome. For example, a guide RNA and Cas
Plants 2021,10, 628 9 of 19
can be expressed transiently in the protoplast to edit a plant genome, and the protoplast can
be regenerated into whole plant. Cas is a class II CRISPR system which is used in various
organisms as a gene editing tool [
138
,
140
]. The basic mechanism involved in CRISPR-
Cas editing is transformation to cells, followed by its integration with the host genome,
and expression, where it cuts the specific locus of interest on the chromosome. The genome
cleavage requires the Cas system, together with a single guided RNA (sgRNA): fusion of
trans-activating (tracr RNA) and CRISPR RNAs (crRNA), followed by the recognition of
the desired DNA sequence and protospacer-adjacent motifs (PAMs) (Figure 3) [138].
Plants 2021, 10, x FOR PEER REVIEW 9 of 19
Cas can be expressed transiently in the protoplast to edit a plant genome, and the proto-
plast can be regenerated into whole plant. Cas is a class II CRISPR system which is used
in various organisms as a gene editing tool [138,140]. The basic mechanism involved in
CRISPR-Cas editing is transformation to cells, followed by its integration with the host
genome, and expression, where it cuts the specific locus of interest on the chromosome.
The genome cleavage requires the Cas system, together with a single guided RNA
(sgRNA): fusion of trans-activating (tracr RNA) and CRISPR RNAs (crRNA), followed by
the recognition of the desired DNA sequence and protospacer-adjacent motifs (PAMs)
(Figure 3) [138].
Figure 3. Illustration of the CRISPR-Cas system for tef genome editing. The gene of interest is
transferred into a binary vector, which will be transferred into the target tissue (e.g., embryogenic
calli) via Agrobacterium transformation, where the CRISPR-Cas protein machinery binds and
breaks the double-stranded DNA of the gene of interest. CRISPR-edited lines will be regenerated
Figure 3.
Illustration of the CRISPR-Cas system for tef genome editing. The gene of interest is
transferred into a binary vector, which will be transferred into the target tissue (e.g., embryogenic
calli) via Agrobacterium transformation, where the CRISPR-Cas protein machinery binds and breaks
the double-stranded DNA of the gene of interest. CRISPR-edited lines will be regenerated from rthe
callus. (Note: the pictures used in this figure were either taken in the author’s labs or drawn using
ChemBioDraw software).
Plants 2021,10, 628 10 of 19
To utilize CRISPR-Cas technology in tef improvement, identification of target genes
that regulate agronomically important traits is crucial. In this review, we explored the draft
genome sequence of tef [
141
] to identify genes that are possible targets for improved yield
and abiotic stress tolerance. We reviewed the literature for genes which are negative regu-
lators of abiotic stress tolerance, and those that regulate plant height and yield attributes
in monocots, including rice, maize, wheat and finger millet, which is closely related to
tef. We then searched for homologs in tef (Table 1) from the Ensembl plant database using
CoGeBlast-comparative genomics databases [
142
]. The tef homologs were aligned with
those in other monocots using the Mega X clustlaw alignment tool [
143
,
144
]. After align-
ment, a phylogenetic tree was constructed using the Mega X maximum likelihood tool [
144
]
(
Figure 4
). It can be observed from Figure 4that the tef homologs showed maximum
bootstrap values with those of the other monocots.
Plants 2021, 10, x FOR PEER REVIEW 10 of 19
from rthe callus. (Note: the pictures used in this figure were either taken in the author’s labs or
drawn using ChemBioDraw software).
To utilize CRISPR-Cas technology in tef improvement, identification of target genes
that regulate agronomically important traits is crucial. In this review, we explored the
draft genome sequence of tef [141] to identify genes that are possible targets for improved
yield and abiotic stress tolerance. We reviewed the literature for genes which are negative
regulators of abiotic stress tolerance, and those that regulate plant height and yield attrib-
utes in monocots, including rice, maize, wheat and finger millet, which is closely related
to tef. We then searched for homologs in tef (Table 1) from the Ensembl plant database
using CoGeBlast-comparative genomics databases [142]. The tef homologs were aligned
with those in other monocots using the Mega X clustlaw alignment tool [143,144]. After
alignment, a phylogenetic tree was constructed using the Mega X maximum likelihood tool
[144] (Figure 4). It can be observed from Figure 4 that the tef homologs showed maximum
bootstrap values with those of the other monocots.
Figure 4. Phylogenetic tree of stress-resistant genes in tef and related monocots. The tree was con-
structed by using specific gene sequences downloaded from NCBI and Ensembl Plants. Bootstrap
values (1000 pseudoreplicates) are shown on the nodes of the branches.
Tef is tolerant to poor soil conditions including waterlogging and drought [145].
However, tef yield is reduced by lodging, terminal drought and diseases. Therefore, tef is
expected to benefit from CRISPR-Cas genome editing technology. The draft genome se-
quence of tef has been released [141]. Two complete homologous chromosomes with
syntenic gene pairs have been reported in the tef genome due to its allotetraploid genome.
The subgenomes are small (~300 Mb), with a low number of transposable elements (TE)
Figure 4.
Phylogenetic tree of stress-resistant genes in tef and related monocots. The tree was
constructed by using specific gene sequences downloaded from NCBI and Ensembl Plants. Bootstrap
values (1000 pseudoreplicates) are shown on the nodes of the branches.
Tef is tolerant to poor soil conditions including waterlogging and drought [
145
].
However, tef yield is reduced by lodging, terminal drought and diseases. Therefore, tef
is expected to benefit from CRISPR-Cas genome editing technology. The draft genome
sequence of tef has been released [
141
]. Two complete homologous chromosomes with
syntenic gene pairs have been reported in the tef genome due to its allotetraploid genome.
The subgenomes are small (~300 Mb), with a low number of transposable elements (TE)
and a high density of genes as compared to other polyploid grasses [
141
]. One of the major
obstacles for the targeted breeding of tef is the presence of genes in two genomes (AA
and BB: tef is allotetraploid, with 2n = 4x = 40 chromosomes). Gene redundancy poses
Plants 2021,10, 628 11 of 19
a difficulty in mutagenesis for developing lodging-resistant and semi-dwarf varieties [
146
].
This obstacle can be overcome by techniques such as targeted genome engineering and
marker assisted selection. In a plant genome, the majority of genes have variable expression
patterns; therefore, the two sub-genomes are more likely to affect agronomic traits with
different frequencies [
141
,
147
]. To utilize CRISPR-Cas technology in tef improvement,
the identification of target genes that regulate agronomically important traits is crucial.
Table 1.
Summary of genes involved in key agronomic traits of selected crops. Homologs of these genes in tef were
downloaded from the genomic database to identify potential candidate genes for CRISPR-Cas-mediated gene editing in tef.
Gene Plant Name Accession Number Reference
Plant Height
KO2Oryza sativa Japonica AY660664 [148]
GA regulatory factor-like (GRF) mRNA Zea mays KJ466125 [149]
growth-regulating factor 10 (GRF10) Oryza sativa Indica FJ546694 [150]
GA20-oxidase (GA20ox2) Oryza granulata EU179380 [151]
BRI1 Triticum aestivum DQ655711 [152]
Sd-1 (used in green revl) Oryza sativa KP212897.1 [70]
RHT1 Triticum aestivum FN649763 [153]
Number of Tillers and Panicle Branches
OsCKX2 Oryza sativa AB205193.1 [154]
teosinte branched1 (tb1) switchgrass AF131673.2 [155]
GSK2 Oryza sativa XM_015782085 [156]
PYL2 Oryza sativa KJ700410.1
[157]
PYL3, Oryza sativa KJ191278.1
PYL4, Oryza sativa KJ855099.1
PYL5, Oryza sativa KJ855100.1
PYL6 Oryza sativa KJ855101.1
PYL12 Oryza sativa KJ855107.1
monoculm1 MOC1 Oryza sativa Japonica KC700671.1 [158]
Grain Size
G1F1A Oryza sativa GU797949 [159]
Drought Tolerance
GhWRKY33 Gossypium hirsutum KJ825875.1 [160]
WRKY mRNA Triticum aestivum KT865879 [161]
threonine dehydratase mRNA Eleusine coracana MK573864 [162]
OsCDPK7 Oryza sativa Japonica AB042550 [163]
TaWRKY146 Triticum aestivum MF770640.1 [164]
NF-Y18 Oryza sativa Japonica HQ731479 [165]
Arginine decarboxylase (ADC) Oryza sativa Japonica CA754598.1 [166]
CIPK12 Oryza sativa Japonica EU703798 [166]
NF-YB Zea mays NM_001112582 [167]
5. Constraints and Solutions Related to CRISPR-Cas Genome Editing
The stable transfer of the transgene into the target site using CRISPR-Cas during
the transformation process may cause the insertion of plasmid DNA or unwanted genes,
which makes it a genetically modified (GM) crop. This limits the use of CRISPR-Cas
system for sustainable agriculture and biotechnology because in some countries the use of
GMOs is either tightly regulated or totally prohibited [
168
]. Although genetic segregation
is the process by which the foreign DNA can be removed, this is not applicable to some
clonally propagated plants. Moreover, in some countries, CRISPR-Cas products are still
not acceptable because foreign DNA materials are used in the process, although these
foreign materials are removed at the end [
168
]. In plants, DNA-free genome editing
has been conducted using two approaches; these are pre-assembled ribonucleoproteins
Plants 2021,10, 628 12 of 19
(RNPs) [
169
,
170
] and the delivery of a combination of guide RNA and mRNA-encoding
Cas [
43
]. However, CRISPR-Cas RNA transient expression efficiency is low, suggesting
a need for additional optimization. Following this approach, the addition of a protectant
for stabilizing RNA could prove to be a promising strategy [171].
Another major drawback of the CRISPR-Cas system is its non-specificity. In this case,
Cas cleaves DNA at non-target sites that are not complementary of single guide RNA [
172
].
This drawback impedes CRISPR-Cas potential applications, particularly when genome
alteration needs to be precise, as in the case of gene therapy. Off-target sites may not
change plant breeding as much as the chemical and physical alterations used in traditional
breeding procedures, which generate many alterations in plants [
173
]. These off-target
alterations can be removed by performing backcrossing to the original plant. However,
this takes several generations of investigation, and the improvement of the process will
be slow.
In plants, the specificity of the CRISPR-Cas system has been estimated by deliberate
non-target investigation [
174
]. For RNPs, non-target alterations were hardly recognized
by thorough sequencing, indicating that RNPs enhance the specificity of the editing sys-
tem [
172
]. However, no study has been reported on Cas specificity in plants. Several
impartial strategies which include Digenome-seq, high-throughput genome-wide translo-
cation sequencing (HTGTS), genome-wide unbiased identification of double stranded
breaks (DSBs) enabled by sequencing (GUIDE-seq) and breaks labeling, enrichment on
streptavidin, and sequencing (BLESS) have been used to investigate non-specific changes
in human cells [
175
–
178
], and these strategies need to be administered in plants to evaluate
Cas specificity at the genome level. The need for improving its specificity is a major chal-
lenge for CRISPR-Cas genome editing, which requires attention. Various strategies have
been established for improving specificity [
179
], including high-fidelity Cas variants and
the Cas paired nickase strategy [180–182].
6. Conclusions
Climate change and global warming are expected to trigger major abiotic stresses,
which are expected to reduce crop yields and ultimately lead to food shortages in the fore-
seeable future. Since agricultural crops fulfill most of the world’s food supply, it should
be the topmost priority of plant biologists to take concrete measures to cope with climate
change and the forecasted food shortages. Climate change and global warming are mani-
fested by abiotic stress factors that could reduce crop productivity. The goal of this review
was to provide an insight on the potential of advanced tools such as CRISPR-Cas for use by
plant biologists in order to improve stress resilience, modify plant architecture and improve
productivity. Application of this cutting-edge technology in underutilized/orphan crops
such as tef will provide several benefits. It is expected to improve food security in the Horn
of Africa, a region which is very vulnerable to the negative impact of climate change,
and which has been experiencing frequent food insecurity and adding to the global refugee
crisis. It will also enhance the acceptance of tef as a healthy and nutritious grain, which
will play a role in reducing micronutrient deficiency.
Author Contributions:
M.N. and A.L.-O. conceived the review; M.N. wrote the draft of the manuscript;
A.L.-O., M.S., Z.T., G.B., A.L.K. and S.A. contributed and edited the manuscript. All authors have
read and agreed to the published version of the manuscript.
Funding:
This manuscript was support by the University of North Carolina at Greensboro (Grant #
133504 to A.L.-O.).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: All the authors declare no conflict of interest.
Plants 2021,10, 628 13 of 19
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